MicroRNA dysregulation in cow's jejunum, jejunal lymph node, and caecal Peyer's patch during Mycobacterium avium subsp. paratuberculosis infection

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Johne's disease (JD), a contagious granulomatous gastroenteritis with global distribution. This study investigated the expression of miRNAs and their potential regulatory mechanisms in Canadian Holstein cows with different MAP infection status, including 4 infected (MAPI), 5 tolerant to MAP infection (MAPT) and 5 healthy controls (HC).Intestinal tissues, jejunum (JE), jejunal lymph node (JELN) and caecal Peyer's patches (CPP) were collected for miRNA-sequencing, differential expression and functional analysis. A total of 318, 281 and 251 miRNAs were identified in JE, JELN, and CPP, respectively. Out of these, 28, 46, and 24 were highly expressed in JE, JELN and CPP, respectively, including 23 miRNAs highly expressed across all tissues. Their functional enrichment indicates overrepresentation in mostly immune related functions. More differentially expressed (DE) miRNAs were identified in CPP (39, 21 and 11) followed by the JELN (25, 8 and 13) and JE (9, 9 and 15) for the comparisons MAPI vs HC, MAPT vs HC, and MAPI vs MAPT, respectively. Six common DE miRNAs (bta-miR-125a, bta-miR-146a, bta-miR-146b, bta-miR-21-5p, bta-miR-320a, bta-miR-370) (FDR < 0.1) in the JELN and CPP of MAPI cows are implicated in the immune response, suggesting roles in MAP infection. Similarly, more functional annotations were recorded for the CPP (1284) than JELN (377) and JE (four). DE miRNAs in JELN and CPP were enriched in gene ontology (GO) terms (e.g. lymphocyte activation, lymphocyte homeostasis, leukocyte differentiation, regulation of cell differentiation, T cell receptor complex, alpha-beta T cell activation) and KEGG pathways (e.g. T/B cell receptor signaling pathways, Chemokine signaling pathway, Leukocyte transendothelial migration, Th17 cell differentiation, MAPK signaling pathway, etc.) with immune related functions indicative of participation in the regulatory mechanisms of the host immune response to MAP infection. The comparison MAPI vs HC revealed a more pronounced immune response in JELN and CPP indicative of a heightened immune response, but fewer DE miRNAs and less pronounced immune activation for MAPT vs HC indicative of the development of tolerance to MAP presence.MiRNA expression and functional enrichment in JE, JELN, and CPP highlightes tissue-specific regulation of immune pathways and cellular functions during JD. Moreover, largely a different set of DE miRNAs, biological processes and pathways were driving the MAPI and MAPT phenotypes. These findings underscore the complex interplay between miRNAs and MAP in the studied tissues and provide a foundation for further exploration of miRNAs as potential biomarkers for the management of JD.